Improving and elucidating factors regulating black walnut ( Juglans nigra L.) clonal propagation

Micah E Stevens, Purdue University


Black walnut (Juglans nigra L.) is a fine hardwood tree species native to the central hardwood region of the United States. High-quality black walnut timber is highly desirable. Traded in both regional and global markets, it has been used for veneer, and the manufacture of high-end products such as cabinets, furniture, and gunstocks. As a result of its high economic value, black walnut has been commercially cultivated for many years, and breeding programs have generated superior timber genotypes with improved marketable traits. Once elite genotypes were developed, it was quickly recognized that black walnut was recalcitrant to clonal propagation techniques. Current black walnut propagation techniques such as grafting are time and labor intensive, and are less than optimal for commercial clonal black walnut production. Reports on black walnut propagation are limited, and successful protocols were often genotype dependent and difficult to replicate. The inability of black walnut to predictably and reliably produce adventitious roots is often the greatest hurdle to successful propagation protocols. The objective of this research was to develop a reproducible and dependable clonal propagation protocol for black walnut, as well as to study the mechanisms controlling adventitious root formation in black walnut. Clonal propagation systems are ideal to rapidly replicate elite genotypes, assist conservation efforts, and act to complement tree improvement programs. These goals were achieved using a multi-faceted approach. First, an in vitro propagation protocol was developed for elite black walnut genotypes 55 and 189. Nodal cutting explants were aseptically cultured on semi-solid Driver and Kuniyuki walnut (DKW) medium supplemented with 8.9 µM benzyladenine (BA), 0.005 µM indole-3-butryic acid (IBA), 200 mg L-1 casein hydrolysate (CH), 50 mg L-1 adenine hemisulfate (AS), 2 ml L-1 Plant Preservative Mixture™ (PPM), and 4.1 µM meta-topolin. This medium resulted in the greatest shoot growth after 8 weeks for both genotypes, 1.7 and 1.3 cm respectively. Long-term survival and proliferation of microshoots was achieved when nodal segments of in vitro grown shoots were cultured in liquid initiation medium in 3 L polycarbonate Fernbach-style flasks on a rotary shaker (100 rpm) under a 16 h photoperiod at 25°C. Elongated microshoots (5–7 cm in length) were rooted (40%) in a slurry-like medium composed of half-strength DKW medium with 0.11% (w/v) Phytagel™ and coarse vermiculite (2:1, v/v) supplemented with 50 µM IBA for 5 weeks. Rooted microshoots shoots were acclimatized to ambient culture room conditions, but did not survive acclimatization to the greenhouse. Softwood cuttings were used to improve ex vitro propagation and to develop a spatial and temporal timeline of the stages of adventitious root formation. Softwood cuttings (15–20 cm) that were collected from juvenile sources of elite genotypes, dipped for 60 s in 93.2 mM indole-3-butyric acid-potassium salt (K-IBA), and then inserted into a moist medium consisting of 3 perlite: 1 coarse vermiculite (v/v) rooted at a 72% frequency. Rooted cuttings were successfully acclimatized to ambient conditions and continued to grow normally. To visualize anatomical changes during root formation, stems were fixed in formaldehyde, embedded in paraffin, serially sectioned, and stained on sequential days throughout root development. Anatomical analysis revealed adventitious root initials by day 16 and root primordia formation by day 18. Rooted cuttings survived acclimatization to the greenhouse, and continued to grow normally. Using this timeline as a reference for the when and where the stages of root formation were occurring, gene expression assays of genes known to participate in adventitious root formation were conducted. The basal end (1–3 cm) of shoots from juvenile and mature origin, were collected on days 0, 8, 16, and 16; and 26 from mature shoots after treatment with 93.3 K-IBA or deionized water (control) and immediately frozen in liquid nitrogen. Shoots were serially sectioned using a Cryostat microtome, and cortical, phloem fiber, or phloem parenchyma tissues were isolated using a laser capture microdissection system. RNA was extracted from the samples, and quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed to characterize relative changes during root development. Rooting-related genes displayed a distinct, localized expression pattern during depending on treatment and age. These findings elucidated the factors contributing to the recalcitrance of adventitious root formation in this economically valuable hardwood species, and may contribute to the improvement of clonal propagation of elite black walnut timber genotypes.




Pijut, Purdue University.

Subject Area

Genetics|Forestry|Plant sciences

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